Proteins destined for the periplasm or the outer membrane of Escherichia coli are synthesized initially in precursor form with a signal sequence at the amino terminus. This signal targets these precursor proteins to the cellular secretion machinery for translation from the cytoplasm. Three different genes were identified both by conditional mutations (sec) that cause a general block to secretion, and as suppressor (prl) of signal sequence mutations: secY/prlA, secE/prlG, and secA/prlD. Biochemical studies using purified components indicate that these three gene products are both necessary and sufficient to reconstitute the translocation reaction in vitro. Homologues of SecY and SecE have been found in many different eubacteria, archaebacteria, and in eukaryotes as diverse as yeast and mammals. In addition, homologues of key components in the well- characterized signal recognition particle (SRP) have been discovered in E. coli. This striking conservation, together with the universal nature of signal sequences, argues strongly that the basic mechanism of protein translocation has been conserved in all of the biological kingdoms. Accordingly, lessons learned from the analysis of the more experimentally tractable bacterial system will have direct-relevance for cell biologists in general. The role of the prokaryotic SRP in protein secretion is not yet clear. We propose several different genetic approaches to test current hypotheses and probe its function. The prl alleles of the three critical sec genes provide unique experimental tools. We hope to exploit these suppressor to probe the interactions between these critical components, and to continue to analyze their mechanistic role. Finally, outer membrane proteins are translocated from the cytoplasm by the Sec machinery, but we do not understand how they are distinguished from periplasmic proteins for assembly in the outer membrane. Strategies to identify cellular components required for this assembly process have been designed and are being implemented. Work done to date has identified a periplasmic stress regulon that is controlled by a two-component regulatory system, CpxA/R. We think it likely that genes in this regulon may code for important protein folding and targeting factors. Accordingly, we also propose to carry out a molecular characterization of this regulon.